Hyaluronic acid based formulations

ABSTRACT

Disclosed herein are soft tissue fillers, for example, dermal and subdermal fillers, based on low molecular weight hyaluronic acids and pharmaceutically acceptable salts thereof, and methods of manufacturing same.

RELATED APPLICATIONS

This application claims priority to and the benefit of PCT applicationNo. PCT/1B2010/002846, filed Nov. 8, 2010, and PCT application No.PCT/IB2010/003008, filed Nov. 23, 2010, and is a continuation in part ofU.S. patent application Ser. No. 12/393,768, filed Feb. 26, 2009, whichclaims priority to U.S. provisional patent application No. 61/085,956,filed Aug. 4, 2008, U.S. provisional patent application No. 61/087,934,filed Aug. 11, 2008, and U.S. provisional patent application No.61/096,278, filed Sep. 11, 2008, and is a continuation in part of U.S.patent application Ser. No. 12/393,884, filed Feb. 26, 2009, whichclaims priority to U.S. provisional application No. 61/085,956, filed onAug. 4, 2008, U.S. provisional application No. 61/087,934, filed Aug.11, 2008, and U.S. provisional application No. 61/096,278, filedSeptember 11, 2008,the disclosure of each of which is herebyincorporated by reference in its entirety.

The present invention generally relates to injectable soft tissuefillers and more specifically relates to hyaluronic acid-based dermaland subdermal fillers having improved properties.

Skin is composed of the epidermis and the dermis. Below these layerslies the hypodermis, which is not usually classified as a layer of skin.The hypodermis is also commonly referred to as subcutaneous fat layer,or subcutaneous tissue. The outermost epidermis is made up of stratifiedsquamous epithelium with an underlying basement membrane. It contains noblood vessels, and is nourished by diffusion from the dermis. The maintype of cells which make up the epidermis are keratinocytes, withmelanocytes and langerhans cells also present. This layer of skin isresponsible for keeping water in the body and keeping other harmfulchemicals and pathogens out.

The dermis lies below the epidermis and contains a number of structuresincluding blood vessels, nerves, hair follicles, smooth muscle, glandsand lymphatic tissue. The dermis (or corium) is typically 3-5 mm thickand is the major component of human skin. It is composed of a network ofconnective tissue, predominantly collagen fibrils providing support andelastic tissue providing flexibility. The main cell types arefibroblasts, adipocytes (fat storage) and macrophages. The hypodermislies below the dermis. Its purpose is to attach the skin to underlyingbone and muscle as well as supplying it with blood vessels and nerves.It is made up of loose connective tissue and elastin. The main celltypes are fibroblasts, macrophages and adipocytes. The hypodermiscontains 50% of body fat. Fat serves as padding and insulation for thebody. Hyaluronic acid (HA) is a part of the dermis composition and is amajor component of the extra cellular matrix.

Facial aging occurs as the result of several factors: inherent changeswithin the skin, effects of gravity, facial muscles acting on the skin(dynamic lines), soft tissue loss or shift and bone loss and loss oftissue elasticity. The skin ages when the epidermis begins to thin,causing the junction with the dermis to flatten. Collagen decreases as aperson ages and the bundles of collagen, which gives the skin turgor,become looser and lose strength. When the skin loses elasticity, it isless able to resist stretching. Coupled with gravity, muscle pull andtissue changes, the skin begin to wrinkle. Water loss and breakdown ofbonds between cells also reduces the barrier function of the skin, whichcan cause the skin's pore size to increases.

As a person ages, the face loses volume, soft tissue, and fat. Theappearance of jowls and folds are usually caused by the drooping offacial tissues and folding of areas where the muscles below are attachedto the skin. As part of the reduction in soft tissue, the aging faceappears more hollow.

More specifically, in various facial areas, such as forehead, eyes,nose, midface and lower face, changes relating to aging have been welldocumented. In forehead area, the forehead and brow droop over time,which lowers the eyebrows and causes the upper eyelid skin to bunch.Forehead lines appear when one tries to hold the brows and eyelids up tocounteract these changes. It is well known that the eyes are often thefirst facial feature to show signs of aging. Skin changes around theeyes occur earlier than in the rest of the face since the skin isthinner around the eyes. The skin here contains fewer glands and issubjected to constant blinking, squinting, rubbing, and pulling. Themidface ages when the cheeks begin to droop, causing nasolabial folds.Nasolabial folds are the lines that run from the sides of the nose tothe corners of the mouth. These folds have been treated with facialfillers. In the nose area, as a person ages, the nose elongates. Commoncauses of elongation are thinning of the soft tissue and loss ofelasticity, which causes “drooping of the tip” and unmasking of thebone, creating a new hump. In the lower face area, as the face ages,facial tissues descend. This results in the so-called “laugh lines”.

Folds and lines in this area have been treated with facial fillers.Further down on the face, the corners of the mouth may droop and descentof the jowls can create folds often referred to as “marionette” lines.Furthermore, jowls form when the cheeks sag around a fixed point alongthe jaw where the facial muscles attach to the jawbone. The facialmuscles continue down into the neck as a sheet called the platysmamuscle. This muscle often gaps in the center of the neck, creating twobands.

Various injectables have been used for restoring tissue loss in theface. In the past, injectable collagen has been used as a soft-tissuefiller to fill wrinkles, lines and scars on the face. Collagen is anaturally occurring protein that supports various parts of the bodyincluding skin, tendons and ligaments. Fat injections have been used foryears to add volume, fill wrinkles, lines and enhance the lips. Fatinjections involve taking fat from one part of the patient's body(abdomen, thighs or buttocks) and injecting it beneath the facial skin.

Hyaluronic acid (HA), now one of the most commonly used components ofcosmetic dermal fillers, is introduced into aging skin to add volume andminimize wrinkles and lines. Hyaluronic acid is a naturally occurring,water soluble polysaccharide, specifically a glycosaminoglycan, which isa major component of the extra-cellular matrix and is widely distributedin animal tissues. The identical structure of hyaluronic acid in allspecies and tissues makes this polysaccharide an ideal substance for useas a bio-material in health and medicine. Hyaluronic acid is present inmany places in the human body. It gives volume to the skin, shape to theeyes and elasticity to the joints.

Different from animal derived hyaluronic acid, non-animal derivedhyaluronic acid is free from animal proteins. This limits the risk ofanimal based disease transmissions or development of allergic reactionsto animal proteins.

HA, also known as hyaluronan, has excellent biocompatibility and, unlikecollegan, does not require any skin testing before implantation. Inaddition, HA has the ability to bind to large amounts of water, makingit an excellent volumizer of soft tissues.

The development of HA-based fillers which exhibit ideal in vivoproperties as well as ideal surgical usability has proven difficult. Forexample, HA-based fillers that exhibit desirable stability properties invivo, can be so highly viscous that injection through fine gauge needlesis difficult. Conversely, HA-based fillers that are relatively easilyinjected through fine gauge needles often have relatively inferiorstability properties in vivo.

One method to overcome this problem is to have an adequate design ofcrosslinked HA-based fillers. Crosslinked HA is formed by reactinguncrosslinked HA with a crosslinking agent under suitable reactionconditions.

It is generally accepted that HA-based dermal fillers having a highviscosity, for example, those that are highly crosslinked and/or made ofhigh molecular weight HA and/or having a high HA concentration tend tolast longer in the body. Conversely, it is generally accepted thatHA-based dermal fillers having a low viscosity, for example, those thatare more lightly crosslinked and/or made up of low molecular weight HAand/or have a low HA concentration, may have a shorter duration in thebody. Naturally, injection of a high viscosity material through a needleis relatively more difficult, and generally requires a smaller gaugeneedle (for instance, 21G, 23G vs 27G, 30G) than injection of arelatively low viscosity material. It has proven difficult to develop anHA based composition that is both easy to inject through a high gaugeneedle (i.e. thin needle) and which has extended duration in the body.Surprisingly, many of the long lasting, highly injectable compositionsof the present invention include a high percentage of relatively lowmolecular weight HA at relatively low concentrations.

The present invention addresses these and other issues by providing aninjectable HA based dermal filler that has enhanced longevity and isextrudable through a fine needle, thus being more comfortable for thepatient during injection and requiring fewer repeated visits to thephysician.

SUMMARY

The present invention relates to soft tissue fillers, for example,dermal and subdermal fillers, based on hyaluronic acid (HA) andpharmaceutically acceptable salts of HA, for example, sodium hyaluronate(NaHA). In some embodiments of the invention, HA-based compositions areprovided which include a therapeutically effective amount of at leastone anesthetic agent, for example, but not limited to, lidocaine.

The present HA-based compositions are relatively highly viscous at restbut with low viscosity under high shear rate, thus facilitatinginjection even through a fine (i.e. high gauge, e.g. 27G, 30G, 31G, 33G)needle, and have enhanced longevity in the body. The compositions maylast up to four months, six months, 12 months or longer, depending onvarious factors such as where the compositions are introduced in thebody. Methods for preparing such HA-based compositions are also providedas well as products made by such methods.

In a broad aspect of the invention, a soft tissue filler composition isprovided which generally comprises a crosslinked hyaluronic acid (HA)component and an uncrosslinked hyaluronic acid component. Thecrosslinked HA component itself comprises a mixture of a first, lowmolecular weight HA material and a second, higher molecular weight HAmaterial. The first molecular weight HA material has a relatively lowmolecular weight and the second molecular weight HA material has arelatively high molecular weight.

Generally, the crosslinked HA component includes more than 50%, forexample, at least 70%, for example, about 90% by weight of the first,low molecular weight HA.

The uncrosslinked HA component is a relatively high molecular weight HAmaterial, for example, a HA having a molecular weight of at least about1.0MDa, and may be present in the composition in an amount of less thanabout 10%, for example, less than 5.0%, for example, less than about2.0%, for example, less than about 1.0%, for example, about 0.95% w/w.

The crosslinking agent may be any suitable crosslinking agent, but in aparticular embodiment, the crosslinking agent is selected from the groupconsisting of 1,4-butanediol diglycidyl ether (BDDE),1,4-bis(2,3-epoxypropoxy)butane, 1,4-bisglycidyloxybutane,1,2-bis(2,3-epoxypropoxy)ethylene and1-(2,3-epoxypropyI)-2,3-epoxycyclohexane, and 1,4-butanediol diglycidylether.

In some embodiments, the compositions further comprise at least activeagent, for example, an anesthetic agent combined with said crosslinkedHA component.

The crosslinked HA component has a total HA concentration of at least10.0 mg/g. In certain specific embodiments, the crosslinked HA componenthas a total HA concentration of at least about 10.0 mg/g, for example,about 15.0 mg/g, about 17.0 mg/g, about 17.5 mg/g or about 20.0 mg/g, orabout 25.0 mg/g.

Methods of making soft tissue filler compositions are also provided. Inone embodiment, a method of making a soft tissue filler compositiongenerally comprise the steps of providing a crosslinked hyaluronic acid(HA) gel comprising a mixture of a first molecular weight HA materialand a second, different, for example, higher, molecular weight HAmaterial, preparing a separate solution of an uncrosslinked hyaluronicacid of a relatively high molecular weight and combining the crosslinkedgel with a small amount of the uncrosslinked solution. The compositionsmay include at least one anesthetic agent combined with said crosslinkedHA component. The crosslinked HA component has a total HA concentrationof at least 10 mg/g.

In embodiments of the invention including an anesthetic agent, the agentmay comprise lidocaine. In a further embodiment, the amount of theanesthetic agent is present at a concentration between about 0.1% andabout 5.0% by weight of the composition. In still another embodiment,the anesthetic agent is present at a concentration between about 0.2%and about 1.0% by weight of the composition. In one embodiment, theanesthetic agent is lidocaine and is present at a concentration of about0.3% by weight of the composition.

In yet another embodiment, the composition has a complex viscosity ofbetween about 50 Pa*s and about 450 Pa*s, for example, when measured atabout 5 Hz with a rheometer using a cone/plate geometry)(4 cm/2° at 25°C.

In one embodiment, the HA component is a gel, for example, a cohesive,hydrated gel. In one embodiment, the HA component is a crosslinked HAgel having no greater than about 1% to about 10% uncrosslinked HA.

In yet other embodiments, the HA component comprises greater than about10%, for example, greater than about 15%, for example, up to or greaterthan about 20% uncrosslinked HA.

Further described herein is a soft tissue filler composition comprising:a HA component crosslinked with 1,4-butanediol diglycidyl ether (BDDE),said HA component having a degree of crosslinking of less than about 5%,for example, about 2%, and an anesthetic component having aconcentration between about 0.1% and about 5.0% by weight of the softtissue filler composition, wherein the anesthetic is lidocaine.

Further described herein are methods of preparing soft tissue fillercompositions, the methods comprising the steps of: providing a HAcomponent crosslinked with at least one crosslinking agent selected fromthe group consisting of 1,4-butanediol diglycidyl ether (BDDE),1,4-bis(2,3-epoxypropoxy)butane, 1,4-bisglycidyloxybutane,1,2-bis(2,3-epoxypropoxy)ethylene and1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, and 1,4-butanediol diglycidylether or combinations thereof; adjusting the pH of said HA component toan adjusted pH above about 7.2; and adding a solution containing atleast one anesthetic agent to the HA component having the adjusted pH toobtain a HA-based filler composition.

In another embodiment, the composition is sterilized, for example, byautoclaving, to form a sterilized composition and wherein the sterilizedcomposition is stable at ambient temperature for at least about 12months, for example, at least 18 months, at least about 24 months ormore.

In still another embodiment, the adjusted pH is above about 7.5. Inanother embodiment, the method further comprises the step ofhomogenizing the HA component during or after the step of adding thesolution containing the at least one anesthetic agent. In a furtherembodiment, the step of homogenizing comprises subjecting thecomposition to mixing with a controlled shear.

In another embodiment, the step of providing a HA component comprisesproviding dry NaHA material and hydrating the dry NaHA material in analkaline solution to obtain an alkaline, uncrosslinked NaHA gel. In yetanother embodiment, the alkaline NaHA gel has a pH greater than about8.0. In still another embodiment the pH is greater than about 10.

In a further embodiment, the HA component comprises less than about 20%uncrosslinked HA and the crosslinked portion of the HA component has adegree of crosslinking of less than about 6% or less than about 5%.

Further described herein is a soft tissue filler composition comprising:a crosslinked hyaluronic acid (HA) component comprising up to 90% w/w oflow molecular weight HA and no more than about 10% w/w of high molecularweight HA crosslinked with 1,4-butanediol diglycidyl ether (BDDE), saidcrosslinked HA component having a degree of crosslinking of less thanabout 5%, an anesthetic component such as lidocaine having aconcentration between about 0.1% and about 5.0% by weight, and less than5.0% w/w of an uncrosslinked component of high molecular weight HA .

The present invention further provides methods of preparing a softtissue filler composition. In some embodiments, a method is providedcomprising the steps of: providing dry NaHA material and hydrating thedry NaHA material in an alkaline solution to obtain an alkaline,uncrosslinked NaHA gel; crosslinking the NaHA gel with BDDE to form acrosslinked alkaline HA composition with a degree of crosslinking lessthan about 5% and a pH above about 7.2; adding a solution containing0.3% lidocaine HCl to the HA component having the adjusted pH to obtainsaid HA-based filler composition; homogenizing the HA-based fillercomposition thereby forming a homogenized HA-based filler composition;adding an amount of a high molecular weight HA uncrosslinked gel to thecomposition, and sterilizing the homogenized HA-based filler compositionthereby forming a sterilized HA-based filler composition.

Definitions

Certain terms as used in the specification are intended to refer to thefollowing definitions, as detailed below. Where the definition of termsdeparts from the commonly used meaning of the term, applicant intends toutilize the definitions provided below, unless specifically indicated.

Autoclave stable or stable to autoclaving as used herein describes aproduct or composition that is resistant to degradation such that theproduct or composition maintains at least one, and preferably all, ofthe following aspects after effective autoclave sterilization:transparent appearance, pH, extrusion force and/or rheologicalcharacteristics, hyaluronic acid (HA) concentration, sterility,osmolarity, and lidocaine concentration.

The term “about” in the context of numerical values will be readilyunderstood by a person skilled in the art, and preferably means thatspecific values may be modified by +/−10%. As regards endpoints ofranges, the modifier “about” preferably means that the lower endpointmay be reduced by 10% and the upper endpoint increased by 10%. It isalso contemplated that each numerical value or range disclosed in thisapplication can be absolute, i.e. that the modifier “about” can bedeleted.

All numbers herein expressing “molecular weight” of HA are to beunderstood as indicating the weight average molecular weight (Mw) inDaltons.

The molecular weight of HA is calculated from an intrinsic viscositymeasurement using the following Mark Houwink relation:

Intrinsic Viscosity (m³/kg)=9.78×10⁻⁶×Mw^(0.690)

The intrinsic viscosity is measured according to the procedure definedEuropean Pharmacopoeia (HA monograph N° 1472, January/2009).

High molecular weight HA as used herein describes a HA material having amolecular weight of at least about 1.0 million Daltons (mw≧10⁶ Da or 1MDa) to about 4.0 MDa. For example, the high molecular weight HA in thepresent compositions may have a molecular weight in the range about 1.5MDa to about 3.0 MDa, or the high molecular weight HA may have a weightaverage molecular weight of about 2.0 MDa. In another example, the highmolecular weight HA may have a molecular weight of about 3.0 MDa.

Low molecular weight HA as used herein describes a HA material having amolecular weight of less than about 1.0 MDa. Low molecular weight HA canhave a molecular weight of between about 200,000 Da (0.2 MDa) to lessthan 1.0 MDa, for example, between about 300,000 Da (0.3 M Da) to about750,000 Da. (0.75 MDa), up to but not exceeding 0.99 MDa.

Preferably, there is no overlap between the molecular weightdistribution of the low and high molecular weight HA materials.

Preferably, the mixture of the low molecular weight HA and highmolecular weight HA has a bimodal molecular weight distribution. Themixture may also have a multi-modal distribution.

In one aspect of the invention, the compositions comprise HA having ahigh molecular weight component and a low molecular weight component,and the high molecular weight component has a weight average molecularweight at least twice the weight average molecular weight of the lowmolecular weight component.

For example, the molecular weight ratio of the high molecular weight HAto the low molecular weight HA in the composition is at least 2:1.

For example, a composition in accordance with this aspect of theinvention may include a low molecular weight component having a weightaverage molecular weight of about 500,000 Da, and a high molecularweight component having a weight average molecular weight of about, orat least about, 1.0 MDa.

In another example, a composition in accordance with the invention mayinclude a low molecular weight component having a weight averagemolecular weight of about 800,000 Da, and a high molecular weightcomponent having a weight average molecular weight of about, or at leastabout, 1.6 MDa.

In a similar aspect of the invention, methods for making a HA basedcomposition are provided which generally include the steps of selectinga low molecular weight HA material and selecting a high molecular weightHA material having a molecular weight at least twice as high as themolecular weight of the low molecular weight material, combining thesehigh and low molecular weight materials, and crosslinking these combinedmaterials with a suitable crosslinking agent.

Degree of crosslinking as used herein refers to the intermolecularjunctions joining the individual HA polymer molecules, or monomerchains, into a permanent structure, or as disclosed herein the softtissue filler composition. Moreover, degree of crosslinking for purposesof the present disclosure is further defined as the percent weight ratioof the crosslinking agent to HA-monomeric units within the crosslinkedportion of the HA based composition. It is measured by the weight ratioof crosslinker to HA monomers (crosslinker : HA monomers).

Uncrosslinked HA as used herein refers to individual HA polymermolecules that are not crosslinked, or are very lightly crosslinked(very low degree of crosslinking). Uncrosslinked HA generally remainswater soluble.

Cohesive as used herein is the ability of a HA-based composition toretain its shape and resist compression. In some embodiments of theinvention, cohesiveness includes the ability of the gel to absorb atleast one time its weight of water without breaking into small pieces ofgel. Cohesiveness is affected by, among other factors, the molecularweight ratio of the initial uncrosslinked HA, the degree ofcrosslinking, the amount of residual uncrosslinked HA followingcrosslinking or added to the composition.

DETAILED DESCRIPTION

The present disclosure generally relates to soft tissue fillers, forexample, injectable dermal and subdermal fillers, based on hyaluronicacids (HA) and pharmaceutically acceptable salts of HA, for example,sodium hyaluronate (NaHA). In one aspect, HA-based compositionsdescribed herein include a therapeutically effective amount of at leastone anesthetic agent, for example, lidocaine. The present HA-basedcompositions may include at least one anesthetic agent, for example,lidocaine. Methods of making these compositions are also provided.

In one aspect of the invention, the compositions maintain at least oneof, or all of, the following aspects after effective autoclavesterilization and/or prolonged storage: transparent appearance, pH foruse in a patient, extrusion force and/or rheological characteristics, HAconcentration, sterility, osmolarity, and lidocaine concentration.Methods or processes of preparing such HA-based compositions are alsoprovided as well as products made by such methods or processes.

As used herein, hyaluronic acid (HA) can refer to any of its hyaluronatesalts, and includes, but is not limited to, sodium hyaluronate (NaHA),potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate, andcombinations thereof. Both HA and pharmaceutically acceptable saltsthereof can be used in this invention.

Generally, the concentration of HA in the compositions described hereinis preferably at least 10 mg/mL and up to about 40 mg/mL.

In certain embodiments, the HA concentration in the crosslinkedcomponent of the present compositions is no greater than about 25 mg/g,for example, about 20.0 mg/g, for example, about 18.0 mg/g, about 17.0mg/g, about 16.0 mg/g, about 15.0 mg/g, about 13.0 mg/g, about 12.0mg/g, about 11.0 mg/g or about 10.0 mg/g. In one embodiment, the HAconcentration in the crosslinked component is about 17.5 mg/g. Inanother embodiment the HA concentration in the crosslinked component is15.0 mg/g. In another embodiment, the HA concentration in thecrosslinked component is about 25.5 mg/g.

In addition, in embodiments with anesthetics, the concentration of oneor more anesthetics is in an amount effective to mitigate painexperienced upon injection of the composition. The at least one localanesthetic can be selected from the group of ambucaine, amolanone,amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine,bupivacaine, butacaine, butamben, butanilicaine, butethamine,butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine,cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, diperodon,dycyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine,beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine,hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate,levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methylchloride, myrtecaine, naepaine, octacaine, orthocaine, oxethazaine,parethoxycaine, phenacaine, phenol, piperocaine, piridocaine,polidocanol, pramoxine, prilocaine, procaine, propanocaine,proparacaine, propipocaine, propoxycaine, psuedococaine, pyrrocaine,ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine,zolamine, and salts thereof. In one embodiment, the at least oneanesthetic agent is lidocaine, such as in the form of lidocaine HCl. Thecompositions described herein may have a lidocaine concentration ofbetween about 0.1% and about 5% by weight of the composition, forexample, about 0.2% to about 1.0% by weight of the composition. In oneembodiment, the composition has a lidocaine concentration of about 0.3%by weight (w/w %) of the composition. The concentration of lidocaine inthe compositions described herein can be therapeutically effectivemeaning the concentration is adequate to provide a therapeutic benefitwithout inflicting harm to the patient.

Further, a method of preparing a HA-based composition is providedwherein the method comprises providing a precursor composition includinga substantially pH neutral, crosslinked HA-based gel and adjusting thepH of the gel to a pH of greater than about 7.2, for example, about 7.5to about 8.0. The method further comprises the step of combining asolution containing lidocaine, for example in the form of lidocaine HCl,with the slightly alkaline gel after the pH has been so adjusted andobtaining a HA-based composition including lidocaine that is stable toautoclaving.

Another method of preparing a HA-based composition containing aneffective amount of lidocaine, generally comprises the steps of:providing purified NaHA material, for example, in the form of fibers;hydrating the material; and crosslinking the hydrated material with asuitable crosslinking agent to form a crosslinked HA-based gel. Themethod further comprises the steps of neutralizing and swelling the gel,and adding to the gel a solution containing lidocaine, preferably anacidic salt of lidocaine chlorhydrate, to form a HA/lidocaine gel.Further still, the method further comprises homogenizing theHA/lidocaine gel, and adding a small amount of an uncrosslinked HAsolution. The composition is then packaged in syringes for dispensing.The syringes are then sterilized by autoclaving at an effectivetemperature and pressure. In accordance with the present description,the packaged and sterilized HA/lidocaine gels exhibit enhanced stabilityrelative to HA-based compositions including lidocaine which are madeusing conventional methods.

The present products and compositions are considered to be sterile whenexposed to temperatures of at least about 120° C. to about 130° C.and/or pressures of at least about 12 pounds per square inch (PSI) toabout 20 PSI during autoclaving for a period of at least about 1 minuteto about 15 minutes.

The present products and compositions also remain stable when stored forlong periods of time at room temperature. Preferably, the presentcompositions remain stable for a period of at least about two months, orat least about six months, or at least about 9 months, or at least about12 months, or at least about 24 months or at least about 36 months, attemperatures of at least about 25° C. In a specific embodiment, thecompositions are stable at a temperature up to about 45° C. for a periodof at least two months.

The manufacturing process includes, in one embodiment, the initial stepof providing raw HA material in the form of dry HA fibers or powder. Theraw HA material may be HA, its salts and/or mixtures thereof. In apreferred embodiment, the HA material comprises fibers or powder ofNaHA, for example, bacterial-sourced NaHA fibers. In some aspects of thepresent description, the HA material may be animal derived. The HAmaterial may be a combination of raw materials including HA and at leastone other polysaccharide, for example, glycosaminoglycan (GAG).

In a broad aspect of the invention, the HA material of the compositionsmay comprise between about 5% to about 95% low molecular weight HA withthe balance of the HA material including high molecular weight HA.

In one aspect of the invention, the HA material in the compositionsnearly entirely comprises or consists of low molecular weight HA. Insome embodiments, nearly 100% of the HA material in the presentcompositions may be low molecular weight HA as defined above. In otherembodiments, the HA material in the compositions comprises a combinationof relatively high molecular weight HA and relatively low molecularweight HA, as defined above. In certain embodiments, at least 50%, forexample, at least about 70%, for example, about 90% or greater of the HAmaterial in the compositions is low molecular weight HA as definedabove, with the remaining portion of HA being high molecular weight HA.

In one aspect of the invention, the ratio of low molecular weight tohigh molecular weight of the HA in the compositions is at least about 2(w/w≧2), with the low molecular weight HA having a molecular weight ofbetween about 0.20 MDa and no greater than about 0.99 MDa.

It will be appreciated by those of ordinary skill in the art that theselection of high and low molecular weight HA material and theirrelative percentages or ratios is dependent upon the desiredcharacteristics, for example, extrusion force, elastic modulus, viscousmodulus and phase angle expressed as the ratio of viscous modulus toelastic modulus, cohesivity, etc. of the final HA-based product. Foradditional information that may be helpful in understanding this andother aspects of the present disclosure, see Lebreton, U.S. PatentApplication Publication No. 2006/0194758, the entire disclosure of whichis incorporated herein by this reference.

In one embodiment, the pure, dry NaHA fibers are hydrated in an alkalinesolution to produce an uncrosslinked NaHA gel. Any suitable alkalinesolution may be used to hydrate the NaHA in this step, for example, butnot limited to aqueous solutions containing sodium hydroxide (NaOH),potassium hydroxide (KOH), sodium bicarbonate (NaHCO₃), lithiumhydroxide (LiOH), and the like. In another embodiment, the suitablealkaline solution is aqueous solutions containing NaOH. The resultingalkaline gel will have a pH above 7.5. The pH of the resulting alkalinegel can have a pH greater than 9, or a pH greater than 10, or a pHgreater than 12, or a pH greater than 13.

The next step in the manufacturing process involves the step ofcrosslinking the hydrated, alkaline NaHA gel with a suitablecrosslinking agent. The crosslinking agent may be any agent known to besuitable for crosslinking polysaccharides and their derivatives viatheir hydroxyl groups. Suitable crosslinking agents include but are notlimited to, 1,4-butanediol diglycidyl ether (or1,4-bis(2,3-epoxypropoxy)butane or 1,4-bisglycidyloxybutane, all ofwhich are commonly known as BDDE), 1,2-bis(2,3-epoxypropoxy)ethylene and1-(2,3-epoxypropyl)-2,3-epoxycyclohexane. The use of more than onecrosslinking agent or a different crosslinking agent is not excludedfrom the scope of the present disclosure. In one aspect of the presentdisclosure, the HA gels described herein are crosslinked using BDDE.

The step of crosslinking may be carried out using any means known tothose of ordinary skill in the art. Those skilled in the art appreciatehow to optimize conditions of crosslinking according to the nature ofthe HA, and how to carry out crosslinking to an optimized degree.

In another embodiment, the crosslinking of the HA is accomplished duringhydration of the HA fibers, by hydrating the combined high and lowmolecular weight fibers in an alkaline solution containing acrosslinking agent, for example, BDDE.

The degree of crosslinking in the HA component of the presentcompositions is at least about 1% and is up to about 20% BDDE/HA, w/w,for example, between about 4% and about 12%, for example, about 10%, forexample, about 8%, for example, about 6%, for example, about 5%, forexample, about 4%.

The hydrated crosslinked, HA gels may be swollen to obtain the desiredHA concentration. This step can be accomplished by neutralizing thecrosslinked, hydrated HA gel, for example by adding an aqueous solutioncontaining of an acid, such as HCl. The gels are then swelled in aphosphate buffered saline (PBS) solution for a sufficient time and at alow temperature.

The gels may now be purified by conventional means such as, dialysisagainst a phosphate buffer, or alcohol precipitation, to recover thecrosslinked material, to stabilize the pH of the material and to removeany un-reacted crosslinking agent. Additional water or a slightlyalkaline aqueous solution can be added to bring the concentration of theHA in the composition to a desired concentration. In some embodiments,the HA concentration of the crosslinked component of the compositions isadjusted to between about 15 mg/g and about 20 mg/g. For example, the HAconcentration of the crosslinked portion of the compositions may beadjusted to yield an HA concentration of about 15 mg/g, about 17 mg/g orabout 20 mg/g.

The pH of the purified, substantially pH neutral, crosslinked HA gelsare preferably adjusted to cause the gel to become slightly alkalinesuch that the gels have a pH of greater than about 7.2, for example,about 7.5 to about 8.0. This step may be accomplished by any suitablemeans, for example, by adding a suitable amount of dilute NaOH, KOH,NaHCO₃ or LiOH, to the gels or any other alkaline molecule, solutionand/or buffering composition know by one skilled in the art.

In some embodiments, an effective amount of a local anesthetic, forexample, lidocaine, such as lidocaine HCl, is then added to the purifiedNaHA gels. For example, in some embodiments, the lidocaine HCl isprovided in a powder form which is solubilized using water for injection(WFI). The gels are kept neutral with a buffer or by adjustment withdiluted NaOH in order that the final HA/lidocaine composition will havea desired, substantially neutral pH. Preferably, the final HA-basedfiller compositions including lidocaine will have a lidocaineconcentration of between at least about 0.1% and about 5%, for example,about 2% by weight of the composition, or in another example about 0.3%.

After the addition of the lidocaine HCl, or alternatively, during theaddition of the lidocaine HCl, the HA/lidocaine gels, or compositions,are homogenized to create highly homogenous HA/lidocaine gels having adesired consistency and stability. Preferably, the homogenization stepcomprises mixing, stirring, or beating the gels with a controlledshearing force obtaining substantially homogenous mixtures.

The HA/lidocaine compositions described herein display a viscosity whichis dependent on the composition's properties and the presence of atleast one anesthetic agent. The viscosity of the HA/lidocainecomposition can be from about 50 Pa*s to about 450 Pa*s. In otherembodiments, the viscosity can be from about 100 Pa*s to about 400 Pa*s,or about from 150 Pa*s to about 350 Pa*s.

In one embodiment, after homogenizing the HA composition, an amount ofuncrosslinked HA solution or gel is added to the composition.

In this aspect of the invention, the added uncrosslinked HA solution hasa HA concentration of between about 10 mg/g and about 50 mg/g, forexample, about 10 mg/g, about 20 mg/g, about 30 mg/g, about 40 mg/g,about 50 mg/g. The HA in the uncrosslinked solution is a high molecularweight HA, having a molecular weight of at least about 1.0 MDa up toabout 4.0 MDa, for example, a molecular weight of about 1.2

MDa, about 1.4 MDa, about 1.6 MDa, about 1.8 MDa, about 2.0 MDa, about2.2 MDa about 2.4 MDa, about 2.6 MDa, about 2.8 MDa, about 3.0 MDa,about 3.2 MDa, about 3.4 MD about 3.6 MDa, about 3.8 The uncrosslinkedHA solution is added to the crosslinked HA component, to produce adermal filler composition having a final weight percent of between 0.5%and 10% uncrosslinked HA.

For example, HA fibers having a molecular weight of about 2.0 MDa areswollen in a phosphate buffer solution to obtain an uncrosslinked HA gelhaving a HA concentration of about 20 mg/g. This uncrosslinked HA gel isthen is added to the crosslinked HA gel composition during a finalmixing step to obtain a dermal filler composition having anuncrosslinked HA gel of about 1.0% w/w.

After adding the uncrosslinked HA gel to the crosslinked HA gel, thecompositions are introduced into syringes and sterilized. Syringesuseful according to the present description include any syringe known inthe art capable of delivering viscous dermafiller compositions. Thesyringes generally have an internal volume of about 0.4 mL to about 3mL, more preferably between about 0.5 mL and about 1.5 mL or betweenabout 0.8 mL and about 2.5 mL. This internal volume is associated withan internal diameter of the syringe which plays a key role in theextrusion force needed to inject high viscosity dermal fillercompositions. The internal diameters are generally about 4 mm to about 9mm, more preferably from about 4.5 mm to about 6.5 mm or from about 4.5mm to about 8.8 mm. Further, the extrusion force needed to deliver theHA compositions from the syringe is dependent on the needle gauge. Thegauges of needles used generally include gauges between about 18G andabout 40G, more preferably about 25G to about 33G, or from about 25G toabout 30G. A person of ordinary skill in the art can determine thecorrect syringe dimensions and needle gauge required to arrive at aparticular extrusion force requirement.

The extrusion forces displayed by the HA compositions described hereinusing the needle dimensions described above are at an injection speedsthat are comfortable to a patient. Comfortable to a patient is used todefine a rate of injection that does not injure or cause excess pain toa patient upon injection to the soft tissue. One skilled in the art willappreciate that comfortable as used herein includes not only patientcomfort, but also comfort and ability of the physician or medicaltechnician injecting the HA compositions. Although certain extrusionforces may be achievable with the HA compositions of the presentdescription, one skilled in the art understands that high extrusionforces can lead to lack of control during injection and that such lackof control may result in additional pain to the patient. Extrusionforces of the present HA/lidocaine compositions can be from about 5 N toabout 20 N, or more preferably from about 8 N to about 15 N, whenextruded at 13.5 mm/min in a 1.0 mL syringe with a standard needle of30G^(1/2).

Sterilization, as used herein comprises any method known in the art toeffectively kill or eliminate transmissible agents, preferably withoutsubstantially altering of degrading the HA/lidocaine compositions.

One preferable method of sterilization of the filled syringes is byautoclave. Autoclaving can be accomplished by applying a mixture ofheat, pressure and moisture to a sample in need of sterilization. Manydifferent sterilization temperatures, pressures and cycle times can beused for this step. For example, the filled syringes may be sterilizedat a temperature of at least about 120° C. to about 130° C. or greater.Moisture may or may not be utilized. The pressure applied is in someembodiments depending on the temperature used in the sterilizationprocess. The sterilization cycle may be at least about 1 minute to about20 minutes or more.

Another method of sterilization incorporates the use of a gaseousspecies which is known to kill or eliminate transmissible agents.Preferably, ethylene oxide is used as the sterilization gas and is knownin the art to be useful in sterilizing medical devices and products.

A further method of sterilization incorporates the use of an irradiationsource which is known in the art to kill or eliminate transmissibleagents. A beam of irradiation is targeted at the syringe containing theHA composition, and the wavelength of energy kills or eliminates theunwanted transmissible agents. Preferable energy useful include, but isnot limited to ultraviolet (UV) light, gamma irradiation, visible light,microwaves, or any other wavelength or band of wavelengths which killsor eliminates the unwanted transmissible agents, preferably withoutsubstantially altering of degrading the HA composition.

EXAMPLE 1 Manufacture of a Low Molecular Weight Soft Filler of theInvention Including Lidocaine

90% of NaHA fibers or powder having a low molecular weight and 10% ofNaHA fibers or powder having a high molecular weight, (ratio of highmolecular weight to low molecular weight of 2:1) are hydrated in analkaline solution, for example, an aqueous solution containing NaOH. Themixture is mixed at ambient temperature, about 23° C., to form asubstantially homogenous, alkaline HA gel.

A crosslinking agent, BDDE, is diluted in an aqueous solution and addedto the alkaline HA gel. The mixture is homogenized for several minutes.

The resulting crosslinked HA gel mixture is then heated at about 50° C.for about 3 hours. The material is now a highly crosslinked HA/BDDE gel(aspect=solid gel). This crosslinked gel is then neutralized with asuitable acidic solution. The neutralized HA gel is then swollen in aphosphate buffer at a cold temperature, for example a temperature ofabout 5° C., to obtain a highly cohesive HA gel. In this specificexample, the phosphate buffered saline solution containswater-for-injection (WFI), disodium hydrogen phosphate, and sodiumdihydrogen phosphate. When neutralized and swollen, the water absorbedby the crosslinked HA component is in a weight ratio of at least 1:1,and without the gel breaking into pieces.

The swollen HA gel is then mechanical stirred and filled into dialysismembranes and dialyzed against a phosphate buffer. The HA gel is filledinto dialysis membranes and dialyzed against a phosphate buffer for upto several days with regular changes of the bath, in order to remove theun-reacted crosslinker, to stabilize the pH close to neutrality (pH=7.2)and to ensure proper osmolarity of the HA gel. The osmolarity of theresulting HA gel is between about 200 mOsmol and about 400 mOsmol, mostpreferably about 300 mOsmol.

After dialysis, the resulting HA gel has a substantially neutral pH,preferably about 7.2.

Lidocaine chlorhydrate (lidocaine HCl) in powder form is firstsolubilized in WFI and filtered through a 0.2 μm filter. Dilute NaOHsolution is added to the HA gel in order to reach a slightly basic pH(for example, a pH of between about 7.5 and about 8). The lidocaine HClsolution is then added to the slightly basic gel to reach a finaldesired concentration, for example, a concentration of about 0.3% (w/w).The resulting pH of the HA/lidocaine mixture is then about 7 and the HAconcentration is about 24 mg/mL. Mechanical mixing is performed in orderto obtain a proper homogeneity in a standard reactor equipped with anappropriate blender mechanism.

An amount of uncrosslinked HA gel is added to the HA/lidocaine gelmixture. Specifically, high molecular weight HA fibers are swollen in aphosphate buffer solution, in order to obtain a homogeneous viscoelasticgel. This uncrosslinked HA gel is then added to the crosslinkedHA/lidocaine gel (for example, at about 1%, w/w). The resulting gel isthen filled into Ready-to-Fill sterile syringes and autoclaved atsufficient temperatures and pressures for sterilization for at leastabout 1 minute.

After autoclaving, the final HA/lidocaine product is packaged anddistributed to physicians. The autoclaved HA/lidocaine product has aviscosity, cohesivity, and extrusion force that are acceptable. Nodegradation of the HA/lidocaine gel product is found during testing ofthe product after the product has spent several months in storage.

EXAMPLE 2 Manufacture of a Low Molecular Weight Soft Filler of theInvention (without Lidocaine)

0.8 g of predried fibers of sodium hyaluronate (NaHA) of having amolecular weight of about 0.2 MDa is weighed out into a firstreceptacle.

0.2 g of predried fibers of NaHA, of having a molecular weight of about2.4 MDa is weighed out into another receptacle.

The two different grades of NaHA are combined.

In a separate receptacle the chosen crosslinking agent, 1,4-butanedioldiglycidyl ether (BDDE), is diluted in 1% sodium hydroxide solution.

6.8 g of the previously prepared BDDE solution diluted to 1/100 are thenadded to the mixed NaHA fibers, still in the solid state, and themixture is homogenized mechanically with a spatula. The mixture is mixedfor two hours at about 20° C. to for a substantially homogenous,alkaline HA gel.

The mixture is then placed in a warm water bath at 50° C. for 2 to 3hours, with further homogenization after 15 minutes of immersion.

The resulting crosslinked HA polymer is then immersed in a phosphatebuffer (PB) to stabilize the pH.

The swollen crosslinked NaHA polymer is then purified by immersion indifferent baths of phosphate buffer to remove unreacted crosslinkingagent and HA.

Dry HA material having a high molecular weight is hydrated in 1 liter ofwater to obtain an uncrosslinked HA gel. 1% of this HA gel correspondingto the amount in the final composition is mixed into the crosslinked HAgel to provide a dermal filler composition in accordance with thepresent invention.

The hydrogel obtained is then homogenized mechanically to ensure thefinal homogeneity, and packed into syringes which are sterilized in anautoclave.

The gel obtained is a long lasting injectable composition using a finegauge needle (e.g. 30 Gauge or 33 Gauge) to improve nasolabial foldlines on the face.

Although the invention has been described and illustrated with a certaindegree of particularity, it is understood that the present disclosurehas been made only by way of example, and that numerous changes in thecombination and arrangement of parts can be resorted to by those skilledin the art without departing from the scope of the invention, ashereinafter claimed.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Notwithstanding that the numericalranges and parameters setting forth the broad scope of the invention areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical value, however,inherently contains certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

1. A soft tissue filler composition comprising: (1) a crosslinkedhyaluronic acid (HA) component comprising a crosslinked mixture of (i) afirst, low molecular weight HA material having a weight averagemolecular weight of between 0.20 about MDa and about 0.99 MDa and (ii) asecond high molecular weight HA material having a weight averagemolecular weight of between about 1.0 MDa and about 4.0 MDa; wherein theweight average molecular weight of the second, high molecular weight HAmaterial is at least twice that of the first, low molecular weight HAmaterial; wherein the HA materials are hyaluronic acid or one or moresalts thereof; wherein the crosslinked HA component has a HAconcentration of between about 15.0 mg/g and about 20.0 mg/g; andwherein the crosslinking is achieved by use of a crosslinking agent; and(2) an uncrosslinked HA component which is HA or a salt thereof, andwhich has a weight average molecular weight of at least about 1.0 MDaand is present in the composition in an amount of less than about 5.0%w/w
 2. The composition of claim 1 wherein the uncrosslinked HA componenthas a weight average molecular weight of at least about 2.0 MDa.
 3. Thecomposition of claim 1 wherein the weight average molecular weight ofthe first, low molecular weight HA material is at least about 500,000Da.
 4. The composition of claim 1 wherein the weight average molecularweight of the second, high molecular weight HA material is at leastabout 1.0M Da.
 5. The composition of claim 1 wherein the weight averagemolecular weight of the first, low molecular weight HA material is about800,000 Da.
 6. The composition of claim 1 wherein the mixture containsabout 50% by weight of the first, low molecular weight HA material. 7.The composition of claim 1 wherein the mixture contains at least about70% by weight of the first, low molecular weight HA material.
 8. Thecomposition of claim 1 wherein the mixture contains about 90% by weightof the first, low molecular weight HA material.
 9. The composition ofclaim 1 wherein the crosslinking agent is selected from the groupconsisting of 1,4-butanediol diglycidyl ether (BDDE),1,4-bis(2,3-epoxypropoxy)butane, 1,4-bisglycidyloxybutane,1,2-bis(2,3-epoxypropoxy)ethylene and1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, and 1,4-butanediol diglycidylether.
 10. The composition of claim 1 wherein the crosslinked HAcomponent has a HA concentration of about 15.0 mg/g.
 11. The compositionof claim 1 wherein the crosslinked HA component has a HA concentrationof about 17.5 mg/g.
 12. The composition of claim 1 wherein theuncrosslinked HA component is present in an amount of less than about1.0% w/w.
 13. The composition of claim 1 wherein the uncrosslinked HAcomponent has a HA concentration of between about 10.0 mg/g and about50.0 mg/g.
 14. The composition of claim 1 further comprising at leastone anesthetic agent combined with said crosslinked HA component.
 15. Asoft tissue filler composition comprising: a crosslinked hyaluronic acid(HA) component comprising a mixture of a first, low molecular weight HAmaterial and a second, high molecular weight HA material, the materialsbeing hyaluronic acid or salts thereof and being crosslinked with acrosslinking agent, the crosslinked HA component having a HAconcentration of between about 15 mg/g and about 20 mg/g, and themixture containing at least 50% by weight of the low molecular weight HAmaterial; the weight average molecular weight of the second, highmolecular weight HA material being at least twice that of the weightaverage molecular weight of the first, low molecular weight HA material;an uncrosslinked hyaluronic acid component or salt thereof, having aweight average molecular weight of at least about 2.4 M Da and an HAconcentration of between about 10 mg/g and about 50 mg/g; theuncrosslinked HA component being present in an amount of less than about5.0% w/w; and at least one anesthetic agent combined with saidcrosslinked HA component.
 16. The composition of claim 15 wherein themixture contains at least 70% by weight of the first, low molecularweight HA material.
 17. The composition of claim 15 wherein the mixturecontains about 90% by weight of the first, low molecular weight HAmaterial.
 18. A method of making a soft tissue filler compositioncomprising the steps of: preparing a crosslinked hyaluronic acid (HA)gel comprising a mixture of a first, low molecular weight HA materialand a second, high molecular weight HA material, the second, highmolecular weight HA material having a weight average molecular weight ofat least twice that of the first, low molecular weight material, themixture comprising more than about 50% by weight of the first, lowmolecular weight HA material, wherein the HA materials are hyaluronicacid or one or more salts thereof; adjusting the HA concentration in thecrosslinked HA gel to a HA concentration of between about 15.0 mg/g andabout 20.0 mg/g; preparing a solution comprising an uncrosslinked HAmaterial or a salt thereof having a weight average molecular weight ofbetween at least about 1.0 MDa and about 4.0 MDa and a concentration ofbetween about 10 mg/g and about 50 mg/g; and combining the gel with thesolution.
 19. The method of claim 18 wherein the mixture comprises morethan about 70% by weight of the first, low molecular weight HA material.20. The method of claim 18 wherein the mixture comprises more than about90% by weight of the first, low molecular weight HA material.
 21. Themethod of claim 18 further comprising the step of combining at least oneanesthetic agent with the gel.
 22. The method of claim 21 wherein thestep of combining at least one anesthetic agent with the gel isperformed prior to the step of combining the gel with the solution.